There has been growing interest in developing methods to detect and quantify circulating tumor cells from blood. Most types of cancer, such as tumors of the breast, prostate, colon, pancreas, esophagus, stomach, and liver are of epithelial origin. Blood cells, on the other hand, are of mesenchymal origin. This difference facilitates the detection of cancer cells, since epithelial-specific markers can be used to identify putative cancer cells in blood. Blood cells will not bear these epithelial-specific markers since they are not of epithelial origin. The presence of circulating tumor cells can be of clinical value in detecting cancer at an early stage, where surgical intervention can be curative. In addition, the presence and number of circulating tumor cells can be of value in cancer patient staging or prognosis, providing an indicator of which patients will likely develop metastases.
There is now considerable evidence that carcinomas shed neoplastic cells into the circulation. This evidence includes studies employing immunomagnetic separation methods to recover circulating epithelial cells from blood [Racila, E., et al. Proc. Natl. Acad. Sci. USA. (1998) 95:4589-4594; Engel, H., et al. Br. J. Cancer. (1999) 81:1165-1173; Kraeft, S.-K., et al. Clin. Cancer Res. (2000) 6:434-442; Wang, Z.-P., et al. Cancer. (2000) 88:2787-2795; Brandt, B., et al. Int. J. Cancer. (1998) 76:824-828; Bilkenroth, U., et al. Intl J. Cancer. (2001) 92:577-582.] as well as those that probe for tumor-specific mRNA in the blood cells of cancer patients. [Lacroix, J., et al. Int. J. Cancer. (2001) 92:1-8; Ghossein, R., et al. J Clin Oncol. (1995) 13:1195-1200; Soeth, E., et al. Cancer Res. (1997) 57:3106-3110; Laribi, A., et al. European Urology. (2001) 39:65-71; Kruger, W., et al. Transfusion. (2000) 40:1489-1493.] The number of circulating neoplastic cells increases with tumor stage. The ability of these cells to establish distant metastatic foci is unclear. Many circulating tumor cells (CTCs) are apoptotic. [Mehes, G., et al. Amer. J. Pathol. (2001) 159:17-20.] It is likely that the cells circulate for hours or days until they are either trapped in the pulmonary vasculature or die. It is probably the rare, exceptional circulating tumor cell that forms a distant metastatic tumor focus.
The rate of neoplastic cell shedding from a solid tumor is undoubtedly quite low, especially in the early stages of tumor growth. Detecting rare CTCs is technically challenging. The success rate depends upon the patients' clinical stage. Isolating CTCs in late-stage neoplastic disease is easier, as there are more of them. In fact, in severe cases, they can even be evident on a routine peripheral blood smear, without any enrichment whatsoever. [Rodriguez-Salas, N., et al. Acta Cytologica. (2000) 44:237-41.] Not all investigators have found CTCs in stage I (localized) disease. The reason for the conflicting findings can relate to the methodologies that have been employed. Subtle methodologic variables have been described that probably account for past discrepant results. [Kruger, W., et al. Transfusion. (2000) 40:1489-1493; Gala, J.-L., et al. Clin Chem. (1998) 44:472-481; Zippelius, A., et al. Clin. Cancer Res. (2000) 6:2741-2750.] Investigators who have employed novel methods to overcome these obstacles have reported circulating neoplastic cells in patients with localized (non-metastatic) tumors. These novel methods include unique cell separation technologies [Racila, E., et al. Proc. NatL. Acad. Sci. USA. (1998) 95:4589-4594.], combining immunomagnetic enrichment with RT-PCR detection [Kruger, W., et al. Transfusion. (2000) 40:1489-1493.], or highly selective primers for specific types of epithelial cells. [Lacroix, J., et al. Int. J. Cancer. (2001) 92:1-8; Laribi, A., et al. European Urology. (2001) 39:65-71.]
Identifying and quantifying circulating carcinoma cells (malignant cells of epithelial origin) has been a technically challenging undertaking. The number of circulating cancer cells depends upon the tumor load, but it is estimated to be approximately one in 1-10 million leukocytes. Therefore, in a 5 milliliter blood sample, there may be only a handful of tumor cells.
Immunomagnetic cell separations involve attaching antibodies directed to proteins found on epithelial cells to small paramagnetic beads. When the antibody-coated beads are mixed with blood, they will attach to and surround epithelial cells. The test tube is then placed in a strong magnetic field, causing the beads to pellet to one side. After removing the blood, captured cells are retained with the beads. The ability of the method to enrich for circulating carcinoma cells would be improved if the high relative numbers of erythrocytes were not in the way. In addition, the beads can sometimes interfere with downstream analysis of the tumor cells.
The currently available methods for recovering so few cells have insufficient cell yields (recoveries), often precluding rare cell isolation and subsequent analysis. Previous immunomagnetic selection studies for capturing small cell subsets from peripheral blood described highly variable cell recovery rates of 24% [Kruger, W., et al. Transfusion. (2000) 40:1489-1493.], 45% [Martin-Henao, G., et al. Transfusion. (2000) 40:35-43.], 47% [Siewert, C., et al. Recent Results Cancer Res. (2001) 158:51-60.], 51% [Hildebrandt, M., et al. Transfusion. (2000) 40:507-512.], 57% [Martin, V., et al. Exp. Hematology. (1998) 26:252-264.], 60% [Werther, K., et al. J. Immunol. Methods. (2000) 238:133-141.], 69.5% [Despres, D., et al. J Hematotherapy & Stem Cell Res. (2000) 9:557-564.] 70-80% [Zigeuner, R., et al. J. Urology. (2000) 164:1834-1837.], & 84% [Bilkenroth, U., et al. Intl J. Cancer. (2001) 92:577-582.]. The recovery rate is important because it establishes a floor on the detection capability for rare cells. The gold standard cell enrichment technology in the field is immunomagnetic enrichment using ferrofluids. The technology is manufactured by Immunicon Corp., Huntingdon Valley, Pa., and commercialized by Veridex LLC, Warren, N.J. A recent paper (2004) correlated the presence of CTCs with prognosis in breast cancer patients. [Cristofanilli, M., et al. New Engl. J. Med. (2004) 351:781-791.] The Veridex/Immunicon ferrofluid technology is the only FDA-cleared technology for measuring CTCs (as a prognostic indicator in breast cancer). An important limitation of the ferrofluid technology is that it appears to not be effective as a cancer screening test.
According to the inventors of the Immunicon/Veridex technology, the limitation in sensitivity can be related to their positive selection method for CTC isolation. [Allard, W., et al. Clin. Cancer Res. (2004) 10:6897-6904.] CTCs are enriched from blood by virtue of their expression of an epithelial cell surface marker not expressed on red and white blood cells. Most commonly, immunomagnetic beads or ferrofluids are coated with an antibody to EpCAM (epithelial cell adhesion marker), a glycoprotein mediating homophilic attachment of epithelial cells. Positive selection methods have two drawbacks. First, disseminated cancer cells are characterized by a high degree of heterogeneity with respect to surface antigens, mutations, and gene expression. [Klein, C., et al. Lancet. (2002) 360:683-689; Braun, S., et al. Int. J. Cancer. (1999) 84:1-5; Pantel, K., et al. J. Natl. Cancer Instit. (1993) 85:1419-1424.] With regard to EpCAM, recent data from Immunicon/Veridex investigators show that circulating tumor cells express much lower levels of EpCAM than cancer cells in the primary tumor. [Doyle, G., et al. J. Clin. Oncol. (2004) 22:9541.] Since EpCAM mediates intercellular attachment, tumor cells must apparently downregulate EpCAM before detaching from the primary tumor. This limits the utility of this widely-used marker for separating tumor cells from blood. Veridex has tried to address this issue by increasing the magnetic load on low-expressing cells. [Liberti, P., et al. J Magnetism Magnetic Materials. (2001) 225:301-307.] Another limiting factor is that many cells often begin to undergo apoptosis after detaching from the primary. [Mehes, G., et al. Amer. J. Pathol. (2001) 159:17-20.] Although they can still be identified by intracellular cytokeratin, cell surface protein expression will decrease as a result.
The overwhelming preponderance of cells in blood are erythrocytes, also known as red blood cells. The two methods of separating white and red blood cells from each other are density gradient sedimentation and chemical lysis. These methods depend upon physical differences between erythrocytes and nucleated blood cells. For isolating CTCs, some believe that density gradient centrifugation is better [Sabile, A., et al. Amer. J. Clin. Pathol. (1999) 112:171-178.] whereas others argue that lysis is better. [Pachmann, K., et al. Clin. Chem. Lab. Med. (2005) 43:617-627.] Neither method is sufficiently reproducible for a CTC clinical test. There are no FDA-cleared tests for CTCs using either method.
Cell separation by density gradient sedimentation relies on a gross physical distinction, cellular density for separating nucleated cells such as CTCs and erythrocytes. Density gradient sedimentation uses media of defined density, such as Percoll or Ficoll, to separate red blood cells from other nucleated blood cells. Lymphocytes and granulocytes are buoyant on a medium of 1.077 g/ml whereas red blood cells sediment. Cultured tumor cells generally are mostly buoyant on a 1.077 g/ml density cushion, but no one has measured the density distribution of actual CTCs isolated from blood. Many blood-borne CTCs are undergoing apoptosis [Mehes, G., et al. Amer. J. Pathol. (2001) 159:17-20.], a factor likely to increase their cellular density. Consequently, there are significant losses associated with density gradient sedimentation. [Choesmel, V., et al. Cancer. (2004) 101:693-703.]
At present an acceptable level of reproducible performance (>80% recovery) with density gradients cannot be obtained. Typically only 40-50% of the starting cells are recovered. Losses occur for a variety of reasons, including the fact that some cells stick to the side of the test tube, at the interface, with a clump of red blood cells, or that some tumor cells sediment with erythrocytes. [Pachmann, K., et al. Clin. Chem. Lab. Med. (2005) 43:617-627.] This can possibly correlate with cell cycle, degree of apoptosis, or other unidentified factors. Other reasons include the fact that the interface is difficult to see when there are few cells, resulting in cellular losses during collection, and/or that the interface is disturbed once someone places a pipette tip in the tube and starts collecting the cells. This agitation disrupts the interface, dispersing the cells and reducing cell recovery. Some cells are also lost in the subsequent centrifugation step (after the density gradient step). The subsequent centrifugation step is for washing out the Percoll or other density media.
Certain chemical solutions (e.g., 150 mM ammonium chloride) are capable of lysing erythrocytes without substantially affecting the viability of white blood cells. Once the red blood cells are lysed, the remaining cells are sedimented by centrifugation. This technique has not been a popular one for enriching CTCs. Exposure to the lysing agent must be carefully controlled, lest nucleated cells also lyse. There are no data on the differential susceptibility of CTCs to lysing agents. Also, the process releases a massive amount of hemoglobin and red blood cells ghosts, both of which interfere with cell separation and downstream analysis.
Another way to remove unwanted cells is by a technique called “panning”. An antibody to the cell type in question is allowed to adhere to a surface, such as the surface of a plastic Petri dish. When the cell mixture is layered on top of the antibody-coated surface, the targeted cells tend to tightly adhere because of the antibody-antigen reaction. Non-adherent cells are rinsed off the surface, thereby effecting a cell separation. Cells that express a cell surface protein recognized by the antibody are retained on the plastic surface whereas other cell types are not. There are two problems with this approach. First, if the red blood cells were to be removed by panning, then a large surface would be needed. There are so many red blood cells in a 5 milliliter blood sample that the surface would have to be quite large, many square meters, to physically accommodate them. In addition, tumor cells sometimes stick non-specifically to plastic surfaces at the interface of density gradients, resulting in their loss.